There are excellent methods available for separating superhelical DNA (covalently closed rings) from a mixture of open circular DNA (rings with one or more single-strand interruptions) and linear DNA (1,2). The separation of open circular DNA from linear DNA presents a more difficult problem. While circular DNA sediments only 13-14s faster than linear D??A of the same molecular weight (3,4), it is difficult to separate these species, especially if the molecules are heterogeneous with respect t#o molecular weight. The studies of ring structures formed after the resection of eukaryotic DNA fragments with exonuclease III and annealing (11) have necessitated the development of an efficient, rapid technique for ring isolation. The repetitive sequences within the rings could then be studied more effectively.
If open circular DNA is to be separated efficiently from linear DNA, it is necessary to capitalize on its circular character rather than on its hydrodynamic properties. Fuke and Thomas (5) attempted such a separation using the technique of agar fixation originally suggested by Wada and Kishizaki (6). They demonstrated that when circular lambda DNA and linear T7 DNA were gelled together with an agar solution, only the T7 could be removed by diffusion. Unfortunately, the technique achieved only partial separation, and was time consuming, requiring several days.
We have used these ideas as a basis for a more efficient separation. Circular and linear DNA are solidified in an agarose gel and the linears are removed preferentially by electrophoresis. The circular DNA probably remains because it' is topologically linked within the agarose meshwork (Fig. 1).
Wit'h this technique, the open circular @X174 RF11 and linear T7 DNA have been efficiently separated from one another. In addition, a device has been built, "The Ring Machine," which simplifies sample preparation and allows better separation by electrophoresis through a thin (2 mm1 agarose pellicle instead of agarose disc gels.